Highly-expressed lncRNA FOXD2-AS1 in adipose mesenchymal stem cell derived exosomes affects HaCaT cells via regulating miR-185-5p/ROCK2 axis

ABSTRACT The healing of skin wounds is a highly coordinated multi-step process that occurs after trauma including surgical incisions, thermal burns, and chronic ulcers. In this study, the authors investigated lncRNA FOXD2-AS1 function in adipose mesenchymal exosomes from ADMSCs that were successfully extracted. Highly expressed lncRNA FOXD2-AS1 in ADMSCs-exosomes accelerated HaCaT cell migration and proliferation. LncRNA FOXD2-AS1 negatively targeted miR-185-5p, and miR-185-5p negatively targeted ROCK2. Highly expressed lncRNA FOXD2-AS1 in ADMSCs-exosomes promoted HaCaT cell migration and proliferation via down-regulating miR-185-5p and further up-regulating ROCK2. In conclusion, LncRNA FOXD2-AS1 overexpression in ADMSCs derived exosomes might accelerate HaCaT cell migration and proliferation via modulating the miR-185-5p/ROCK2 axis.


Introduction
The healing of skin wounds is a highly coordinated multi-step process that occurs after trauma, including surgical incisions, thermal burns, and chronic ulcers [1]. The failure of proceeding through such orderly and timely reparation can induce chronic nonhealing wounds including diabetic, venous, and decubitus skin ulcers [2,3]. Refractory wound is a huge burden to both the patient and society. Thus, exploring a novel marker for wound healing therapy and clarifying the mechanisms of this fatal disease are imperative.
Mesenchymal stem cells (MSCs) are one type of multipotent progenitor cells that are derived from bone marrow, umbilical cord, and adipose tissue and play a well-known function in tissue regeneration [4]. In recent years, more and more studies have strongly proved that exosomes derived from adipose-derived mesenchymal stem cells (ADMSCs) are safe and have become the hot pot of many researches in many different fields, such as wound healing [5][6][7]. A previous study has demonstrated that ADMSCs-derived exosomes can accelerate cell proliferation and migration through regulating Wnt/β-catenin pathway in cutaneous wound healing [8]. ADMSCs-derived exosomes are reported to promote wound healing via accelerating keratinocyte migration and proliferation [9]. Moreover, human ADMSCs are an attractive resource for wound healing due to their regenerative ability to promote injury repair [10]. A study has confirmed that ADMSCs could accelerate wound healing through optimizing fibroblasts characteristics [11], which further verified their regenerative ability to promote injury repair in wound healing.
Long noncoding RNAs (lncRNAs) have been reported to exist in exosomes and modulate gene expression in host cells through intercellular communication [12]. For example, the high expression of lncRNA H19 in ADMSCs-exosomes can up-regulate the expression of SOX9 through miR-19b to promote wound healing [13]. A study has proven that exosomal lncRNA FOXD2-AS1 can act as the promising biomarkers for the diagnostics of colorectal cancer [14]. LncRNA FOXD2-AS1 promotes the progression of a variety of tumours [15][16][17][18]. Moreover, lncRNA FOXD2-AS1 promotes the proliferation of many kinds of cells, including trophoblast cell [19], fibroblast-like synoviocytes [20], and chondrocyte [21]. However, the evidence regarding the implication of exosomal lncRNA FOXD2-AS1 derived from ADMSCs in wound healing is lacking.
In the current study, we paid attention to evaluate the effect of exosomal lncRNA FOXD2-AS1 derived from ADMSCs in wound healing and its potential mechanisms. Our findings demonstrated that highly expressed lncRNA FOXD2-AS1 in ADMSCs derived exosomes accelerated HaCaT cell migration and proliferation via the regulation of miR-185-5p/rho-associated coiled-coil-containing protein kinase 2 (ROCK2) axis, suggesting that exosomal lncRNA FOXD2-AS1 derived from ADMSCs may be a novel therapeutic strategy for wound healing.

Identification of ADMSCs and exosomes
Flow cytometric analysis was utilized to examine the cell surface protein expression of isolated ADMSCs. As shown in Figure 1a, ADMSCs were positive with CD44 and CD105, but negative with CD31 and HLA-DR. Then, we further identified ADMSCs-exomoses through TEM (Figure 1b). Furthermore, the data of NTA confirmed that the particle size of the extracted exosomes was mainly distributed at about 100 nm ( Figure 1c). Moreover, the presentation of specific biomarkers CD9, CD63 and TSG101 indicated that the exosomes were successfully extracted (Figurerue 1D).

miR-185-5p negatively targeted ROCK2
As shown in Figure 6a, the expression of ROCK2 was upregulated in HaCaT cell followed by transfection of ROCK2 overexpression vector. Additionally, the expression of ROCK2 was downregulated followed by transfection of miR-185-5p mimics (Figure 6b). Figure 6c is predicted for binding sites between miR-185-5p and ROCK2. Figure 6d proves miR-185-5p mimics decreased luciferase activity in the ROCK2-WT group, while had no function on luciferase activities in ROCK2-MUT group. Furthermore, Figure 5e and 5f data showed that ROCK2 expression were higher in mimics NC + ROCK2 vector group than that in mimics NC + NC vector group, but lower in miR-185-5p mimics + NC vector group. Meanwhile, mRNA and protein expression of ROCK2 in miR-185-5p mimics + ROCK2 vector group was reduced relative to mimics NC + ROCK2 vector group, but higher than that in miR-185-5p mimics + NC vector group (Figure 6e and 6f). The above data suggested that miR-185-5p could negatively target ROCK2. Moreover, we also found that exosomes treatment significantly induced ROCK2 expression, and lncRNA FOXD2-AS1 overexpressiontransfected exosomes further promoted the expression of ROCK2 (Figure 6g and 6h).

miR-185-5p overexpression inhibited HaCaT cell migration and proliferation through down-regulating ROCK2
As shown in Figure 7a and 7b, the proliferation of HaCaT cells was notably elevated in mimics NC + ROCK2 vector group than that in mimics NC + NC vector group, but downregulated in miR-185-5p mimics + NC vector group. HaCaT cell proliferation in miR-185-5p mimics + ROCK2 vector group was decreased compared with mimics NC + ROCK2 vector group but was increased compared with miR-185-5p mimics + NC vector group (Figure 7a and 7b). In comparison with mimics NC + NC vector group, HaCaT cell migration was promoted in mimics NC + ROCK2 vector group and decreased in miR-185-5p mimics + NC vector group (Figure 7c). Meanwhile, HaCaT cell migration in miR-185-5p mimics + ROCK2 vector group was inhibited relative to mimics NC + ROCK2 vector group, but was elevated relative to miR-185-5p mimics + NC vector group (Figure 7c). Moreover, western blot results confirmed that MMP-2 and MMP-9 level was higher in mimics NC + ROCK2 vector group than those in mimics NC + NC vector group, but lower in miR-185-5p mimics + NC vector group (Figure 7d). Meanwhile, MMP-2 and MMP-9 level in miR-185-5p mimics + ROCK2 vector group was decreased when compared with mimics NC + ROCK2 vector group, and increased in miR-185-5p mimics + NC vector group (Figure 7d).

Discussion
A basic characteristic of wound healing is the repair of the intact epidermal barrier via reepithelialization [22]. It is reported that keratinocytes proliferation and migration are closely related to reepithelialization and closure of the wound gap [23,24]. In the current study, highly expressed lncRNA FOXD2-AS1 in ADMSCs derived exosomes promoted HaCaT cell migration and proliferation via modulating the miR-185-5p/ ROCK2 axis.
Currently, increasing evidences have suggested that the novel role of exosomes as cell communication bodies is dependent on exosomal cargo, including lncRNAs, microRNAs (miRNAs) and proteins [25,26]. Furthermore, accumulating evidences relate regulatory lncRNAs to human diseases. LncRNA FOXD2-AS1 may accelerate proliferation and migration activities in a various of cells [20,27,28]. Interestingly, a study has proven that exosomal lncRNA FOXD2-AS1 can also act as the promising biomarkers for the diagnostics of colorectal cancer [14]. Therefore, we explored the effects of exosomal lncRNA FOXD2-AS1 derived from ADMSCs in wound healing. Our data demonstrate that highly expressed lncRNA FOXD2-AS1 in ADMSCs-exosomes might accelerate HaCaT cell migration and proliferation.
Recent research data suggest that miRNAs are promising tools for the treatment and diagnosis of skin wound healing because miRNAs are important regulators of cellular physiology and pathology [29]. miR-200b/c-3p can modulate epithelial plasticity and repress cutaneous wound healing through the regulation of TGF-βmediated RAC1 pathway [30]. It is interesting that lncRNA interacts with miRNA and the interactions play critical roles in determining cell fate [31]. Adiposederived stem cells-exosomes containing lncRNA MALAT1 can promote wound healing via targeting miR-124 [25]. LncRNA GAS5 is proved to accelerate diabetic wound healing and promote lymphangiogenesis by miR-217/Prox1 axis [32]. Through bioinformatics analyses and Dual-luciferase reporter assay, we verified that lncRNA FOXD2-AS1 targetly regulates miR-185-5p. In addition, lncRNA FOXD2-AS1 directly interacts with miR-185-5p as miRNA sponge and promotes tumour progression, including colorectal cancer [33], papillary thyroid cancer [34], and glioma [35]. In the current study, lncRNA FOXD2-AS1 overexpression in ADMSCsexosomes promoted the migration and proliferation of HaCaT cells via down-regulating miR-185-5p.
MiRNAs are verified to reverse gene expression via degrading mRNA and inhibiting translation [36]. ROCK2 is widely involved in cell biological activities and plays an important role in controlling various cell phenomena [37]. ROCK2 has been suggested to negatively regulate the Parkin-dependent mitophagy pathway [38]. ROCK2 contributes to diabetes-induced impaired cardiac Ca 2+ homoeostasis [39]. ROCK2induced glycolysis and proliferation in osteosarcoma [40]. ROCK2 promotes osteosarcoma growth and metastasis by modifying the PFKFB3 ubiquitination and degradation [37]. ROCK2 promotes invasion and metastasis in hepatocellular carcinoma through disturbing MKP1 [41]. Croze et al. have reported that the suppression of ROCK can promote attachment, proliferation, and wound closure in human embryonic stem cell-derived retinal pigmented epithelium [42]. Additionally, miR-203 overexpression inhibits the proliferation of epidermal stem cells through the downregulation of ROCK2 associated with Notch and Wnt pathways, leading to a delayed wound healing [43]. We verified miR-185-5p can targetly regulate ROCK2 in this study. In addition, Niu et al. have proved that miR-185-5p may inhibit hepatocellular carcinoma cell migration through targeting ROCK2 [44]. In the present study, we demonstrated that miR-185-5p overexpression could inhibit the migration and proliferation of HaCaT cells through down-regulating ROCK2.

Conclusion
In the current study, highly expressed lncRNA FOXD2-AS1 in ADMSCs derived exosomes might accelerate HaCaT cell migration and proliferation via modulating the miR-185-5p/ROCK2 axis, revealing that exosomal lncRNA FOXD2-AS1 derived from ADMSCs may be a novel therapeutic strategy for wound healing.

Isolation and identification of human ADMSCs
The adipose tissues were acquired from the discarded tissue of a 5-year-old female patient who underwent full-layer abdominal skin transplant surgery in our hospital. The protocol of this research has been approved by the Ethics Committee of Weifang Medical University. All patients have signed written informed consent. First of all, adipose tissues were cut into cubes of about 1 mm 3 , which were free of fibre or blood vesicles. Following washed using 0.9% NS, adipose tissues were digested applying 0.75% type I collagenase at 37°C for 1 h. Subsequently, the resuspended cells were transplanted into DMEM medium (Gibco, USA). The 3 rd ADMSCs were used for the subsequent experiment. ADMSCs characterization was evaluated utilizing flow cytometry, as described previously [45]. The antibodies for CD44 (1:100; #ab243894), CD105 (1:200; #ab231774), CD31 (1:200; #ab9498), and HLA-DR (1:100; #ab20181) were obtained from Abcam.

Isolation and identification of exosomes
At 48 h posttransfection, exosome was collected applying the Ribo™ Exosome Isolation Reagent (Ribobio, Guangzhou, China). The extracted exosome morphology was determined using transmission electron microscopy (TEM; Leica, Germany). Exosome size distribution was analysed using the nanoparticle tracking analysis (NTA) as previously described [46]. Finally, the expression of CD9, CD63 and TSG101 was evaluated by western blotting.

Cell counting kit-8 (CCK-8) assay
The cell proliferation was evaluated by a CCK-8 Cell Proliferation and Cytotoxicity Assay Kit (DOJINDO, Japan). HaCaT cells (1 × 10 4 cells/well) were cultured in a 96-well plate. CCK-8 solution (10 μL) was added into plate, subsequently the cells were further incubated for 48 h at 37°C with 5% CO 2 . Optical density at 450 nm was analysed applying a microplate reader.

5-ethynyl-20-deoxyuridine (EdU) assay
The rates of the proliferating cells were determined with an EdU proliferation kit (Beyotime, Shanghai, China). HaCaT cells (1 × 10 4 cells/well) were incubated in a 96-well plate, and the medium with EdU solution (50 μM) was added. Following 120 min of culture, the 4′, 6-diamidino-2-phenylindole (DAPI, Beyotime) was added into each well of the plates and shielded from light for 30 min. Finally, the EdU-positive cells were visualized using a fluorescent microscope and quantified applying ImageJ software.

Wound healing assay
HaCaT cells (4 × 10 4 cells/well) were plated into a 96well plate and cultured till reaching 90% confluence. After that, sterile pipette tips were applied to scrape cell confluent monolayer in a standardized manner, creating a cell-free zone in each well. After the culture of HaCaT cells for 48 h at 37°C, the wound's distance at 0, 24, and 48 h was photographed under the light microscope.

Statistical analysis
Data obtained in the study were represented as the mean ± SD of three independent experimental repeats and measured utilizing GraphPad Prism 8.0. Statistical differences were determined by Student's t-test or oneway ANOVA. A p-value <0.05 was considered to be significant in all experiments.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The author(s) reported that there is no funding associated with the work featured in this article.

Ethics approval and consent to participate
The protocol of this research has been approved by the Ethics Committee of Weifang Medical University. (WFYXY20201003) All patients have signed written informed consent. The experiments were carried out according to the principles of the Declaration of Helsinki.

Consent for publication
All authors have read and approved the manuscript.

Data Availability statement
The datasets used and analysed during the current study are available from the corresponding author on reasonable request.

Author contributions
Conception and design: Huanchao Chang; Perform research: Huanchao Chang, Junliang Chen, and Tianling Cheng; Data analysis and interpretation: Kun Ding and Shengjian Tang; Manuscript writing: All authors; Final approval of manuscript: All authors